Study On Isolation And Co-culture Of Human Adipose-derived Stem Cells And Rabbit Corneal Endothelial Cells In Vitro

Objective: The main aim of this paper is that we cutured human adipose-derivedstem cells(ADSCS)and rabbit corneal endothelial cells and differentiated hADSCSintocorneal endothelial-like cell in vitro,then detected the exptession of its functionalprotein-Aquaporin1to explored that if the differentiated ADSCScould be the alternativeto endothelial keratoplasty.Methods：1.We got the human abdominal adipose from the hospital operating room andextracted the mesenchymal stem cells from it.We described the state of the cells growthand identify the surface antigen of the3rd generation cells by flow cytometry.2.Descemetmembrane and endothelial cell layer were isolated through themicroscope,then dissected and seeded in culture container.We described the cornealendothelial cells(CECS)growth state and identify its relatively specific proteinnerve-spencific enolase(NSE)by immunohistochemistry.3.Transwell inserts containing CECSwere put in6-well plates containing hADSCSto make up the coculture system. CECSplated in the upper compartment.Observed themorphology of induced hADSCSand identify the exptession of AQP1by immunohistochemistry.Results:1.The primary hADSCSshowed typical fibroblast-like after7~9days cultured invitro and covered80%of the bottom after12~14days cultured.The passage cellsproliferated significantly faster than the primary cells.The cells growth curve was"S"shape,and showed that the3~5days was logarithmic growth phase.Flow cytometryresult showed that positive ratecell of surface antigen expression of CD44was93.7%,CD341.6%. 2.After5d cultured,the primary rabbit corneal endothelial cells formed a singlelayer and was "paving stones"shape.Cells was awirling arrangement and central cellswas triangular or short spindle in shape10days later.Passaged cells was hexagogal orpolygona.The cells growth curve was"S"shape,and showed that the2~5days waslogarithmic growth phase.Cells’ expression of NSE was positive by immuno histochemistry.3.CECSand hADSCScultured for10days in transwell co-culture system.Cells’morphology had no significant change.Cells’ expression of AQP1was positive byimmunohistochemistry.Conclusion:Through above methods we could obtain activated、pure hADSCSandCECS;Useing transwell co-culture system can successfully induced hADSCSexpress thecorneal endothelial cells’ function protein AQP1;Induced hADSCSmay be the seed cellsfor transpiantation to treat corneal endothelial disease and injury.